Microdroplet reactors for high-throughput chemistry and biology

Droplet-based microfluidic systems have recently been developed to overcome the problems of slow mixing and dispersion associated with traditional microfluidic systems. By utilising flow instabilities between two immiscible phases, droplets can be generated using normal microfluidic formats. Further, aqueous solutions can be confined and mixed within droplets, resulting in rapid homogenisation and no dispersion. Accordingly, droplet-based microfluidic systems have been utilised in various applications in a high-throughput manner. However, the techniques and methods for droplet formation, manipulation and detection have been continuously studied and improved upon to develop, prepare, manipulate and implement droplet systems for real-world applications. Since droplets can be controllably produced with variable reagent compositions at high generation frequencies (1 kHz or above), on-line detection and characterisation of every high-speed droplet is one of the most important challenges associated with droplet analysis. The ability to extract information from each droplet microreactor is crucial for applications in high-throughput analysis and screening. An appropriate detection technique able to extract the vast amount of information produced in such systems is key in unlocking the full capabilities of droplet-based. In this work, a custom built confocal spectroscopic system was coupled with a droplet-based microfluidic system to conduct high-sensitivity and high-throughput biological experiments. The integration of a confocal system allows for online characterisation of individual droplets in terms of their size, formation frequency, fluorescence intensity and population. The combination of a droplet-based microfluidic system and the confocal detection setup has been successfully used to demonstrate a few high-throughput chemical and biological applications. For example, the droplet system was utilised to demonstrate high-throughput single cell encapsulation, characterisation and quantification for the first time. In addition, highthroughput binding assays and kinetic measurements using a well-known streptavidin-biotin binding model and a protein-protein interaction were performed. Furthermore, a novel approach for fluorescence lifetime imaging (FLIM) was developed and used to analyse mixing patterns within droplets. Specifically, data from FLIM measurements were extracted to determine spatially localised fluorescence lifetimes within droplets and thus a twodimensional map of droplet mixing. Finally, the droplet-based microfluidic approach was exploited to perform biological analysis at the single molecule level

RNA–protein binding kinetics in an automated microfluidic reactor

Microfluidic chips can automate biochemical assays on the nanoliter scale, which is of considerable utility for RNA–protein binding reactions that would otherwise require large quantities of proteins. Unfortunately, complex reactions involving multiple reactants cannot be prepared in current microfluidic mixer designs, nor is investigation of long-time scale reactions possible. Here, a microfluidic ‘Riboreactor’ has been designed and constructed to facilitate the study of kinetics of RNA–protein complex formation over long time scales. With computer automation, the reactor can prepare binding reactions from any combination of eight reagents, and is optimized to monitor long reaction times. By integrating a two-photon microscope into the microfluidic platform, 5-nl reactions can be observed for longer than 1000 s with single-molecule sensitivity and negligible photobleaching. Using the Riboreactor, RNA–protein binding reactions with a fragment of the bacterial 30S ribosome were prepared in a fully automated fashion and binding rates were consistent with rates obtained from conventional assays. The microfluidic chip successfully combines automation, low sample consumption, ultra-sensitive fluorescence detection and a high degree of reproducibility. The chip should be able to probe complex reaction networks describing the assembly of large multicomponent RNPs such as the ribosome

Measuring proteins in H2O with 2D-IR spectroscopy

The amide I infrared band of proteins is highly sensitive to secondary structure, but studies under physiological conditions are prevented by strong, overlapping water absorptions, motivating the widespread use of deuterated solutions. H/D exchange raises fundamental questions regarding the impact of increased mass on protein dynamics, while deuteration is impractical for biomedical or commercial applications of protein IR spectroscopy. We show that 2D-IR spectroscopy can avoid this problem because the 2D-IR amide I signature of proteins dominates that of water even at sub-millimolar protein concentrations. Using equine blood serum as a test system, we investigate the significant implications of being able to measure the spectroscopy and dynamics of proteins in water, demonstrating relevance in areas ranging from fundamental science to the clinic. Measurements of vibrational relaxation dynamics of serum proteins reveals that deuteration slows down the rate of amide I vibrational relaxation by >10%, indicating a dynamic impact of isotopic exchange in some proteins. The unique link between protein secondary structure and 2D-IR amide I lineshape allows differentiation of signals due to albumin and globulin protein fractions in serum leading to measurements of the biomedically-important albumin to globulin ratio (AGR) with an accuracy of ±4% across a clinically-relevant range. Furthermore, we demonstrate that 2D-IR spectroscopy enables differentiation of the structurally similar globulin proteins IgG, IgA and IgM, opening up a straightforward spectroscopic approach to measuring levels of serum proteins that are currently only accessible via biomedical laboratory testing

High sensitivity and label-free oligonucleotides detection using photonic bandgap sensing structures biofunctionalized with molecular beacon probes

A label-free sensor, based on the combination of silicon photonic bandgap (PBG) structures with immobilized molecular beacon (MB) probes, is experimentally developed. Complementary target oligonucleotides are specifically recognized through hybridization with the MB probes on the surface of the sensing structure. This combination of PBG sensing structures and MB probes demonstrates an extremely high sensitivity without the need for complex PCR-based amplification or labelling methods

Microfluidic device for robust generation of two-component liquid-in-air slugs with individually controlled composition

Using liquid slugs as microreactors and microvessels enable precise control over the conditions of their contents on short-time scales for a wide variety of applications. Particularly for screening applications, there is a need for control of slug parameters such as size and composition. We describe a new microfluidic approach for creating slugs in air, each comprising a size and composition that can be selected individually for each slug. Two-component slugs are formed by first metering the desired volume of each reagent, merging the two volumes into an end-to-end slug, and propelling the slug to induce mixing. Volume control is achieved by a novel mechanism: two closed chambers on the chip are initially filled with air, and a valve in each is briefly opened to admit one of the reagents. The pressure of each reagent can be individually selected and determines the amount of air compression, and thus the amount of liquid that is admitted into each chamber. We describe the theory of operation, characterize the slug generation chip, and demonstrate the creation of slugs of different compositions. The use of microvalves in this approach enables robust operation with different liquids, and also enables one to work with extremely small samples, even down to a few slug volumes. The latter is important for applications involving precious reagents such as optimizing the reaction conditions for radiolabeling biological molecules as tracers for positron emission tomography

Microdroplet reactors for high-throughput chemistry and biology

Droplet-based microfluidic systems have recently been developed to overcome the problems of slow mixing and dispersion associated with traditional microfluidic systems. By utilising flow instabilities between two immiscible phases, droplets can be generated using normal microfluidic formats. Further, aqueous solutions can be confined and mixed within droplets, resulting in rapid homogenisation and no dispersion. Accordingly, droplet-based microfluidic systems have been utilised in various applications in a high-throughput manner. However, the techniques and methods for droplet formation, manipulation and detection have been continuously studied and improved upon to develop, prepare, manipulate and implement droplet systems for real-world applications. Since droplets can be controllably produced with variable reagent compositions at high generation frequencies (1 kHz or above), on-line detection and characterisation of every high-speed droplet is one of the most important challenges associated with droplet analysis. The ability to extract information from each droplet microreactor is crucial for applications in high-throughput analysis and screening. An appropriate detection technique able to extract the vast amount of information produced in such systems is key in unlocking the full capabilities of droplet-based. In this work, a custom built confocal spectroscopic system was coupled with a droplet-based microfluidic system to conduct high-sensitivity and high-throughput biological experiments. The integration of a confocal system allows for online characterisation of individual droplets in terms of their size, formation frequency, fluorescence intensity and population. The combination of a droplet-based microfluidic system and the confocal detection setup has been successfully used to demonstrate a few high-throughput chemical and biological applications. For example, the droplet system was utilised to demonstrate high-throughput single cell encapsulation, characterisation and quantification for the first time. In addition, highthroughput binding assays and kinetic measurements using a well-known streptavidin-biotin binding model and a protein-protein interaction were performed. Furthermore, a novel approach for fluorescence lifetime imaging (FLIM) was developed and used to analyse mixing patterns within droplets. Specifically, data from FLIM measurements were extracted to determine spatially localised fluorescence lifetimes within droplets and thus a twodimensional map of droplet mixing. Finally, the droplet-based microfluidic approach was exploited to perform biological analysis at the single molecule level.EThOS - Electronic Theses Online ServiceRoyal Thai GovernmentGBUnited Kingdo

Droplet microfluidics: from proof-of-concept to real-world utility?

Droplet microfluidics constitutes a diverse and practical tool set that enables chemical and biological experiments to be performed at high speed and with enhanced efficiency when compared to conventional instrumentation. Indeed, in recent years, droplet-based microfluidic tools have been used to excellent effect in a range of applications, including materials synthesis, single cell analysis, RNA sequencing, small molecule screening, in vitro diagnostics and tissue engineering. Our 2011 Chemical Communications Highlight Article [Chem. Commun., 2011, 47, 1936–1942] reviewed some of the most important technological developments and applications of droplet microfluidics, and identified key challenges that needed to be addressed in the short term. In the current contribution, we consider the intervening eight years, and assess the contributions that droplet-based microfluidics has made to experimental science in its broadest sense.ISSN:1359-7345ISSN:1364-548